Insulin is a hormone produced in the pancreas by β-cells. The function of insulin is to regulate the amount of glucose (sugar) in the blood, which enters cells through receptors that accept insulin and allow glucose to enter. Once inside, glucose can be used as fuel. Excess glucose is stored in the liver and muscles in a form called glycogen. When blood glucose levels are low, the liver releases glycogen to form glucose. Without insulin, glucose has difficulty entering cells.
In persons with diabetes mellitus, one of the most common metabolic diseases affecting hundreds of millions of individuals worldwide, the pancreas produces no insulin, too little insulin to control blood sugar, or defective insulin. Without insulin, these symptoms progress to dehydration, resulting in low blood volume, increased pulse rate, and dry, flushed, skin. In addition, ketones accumulate in the blood faster than the body is able to eliminate them through the urine or exhaled breath. Respiration becomes rapid, and shallow and breath has a fruity odor. Other symptoms indicating a progression towards diabetic ketoacidotic coma (DKA) include vomiting, stomach pains, and a decreased level of consciousness. The disease leads to serious complications, including hyperglycemia, macroangiopathy, microangiopathy, neuropathy, nephropathy and retinopathy. As a result, diabetes adversely affects the quality of life.
There are two forms of diabetes mellitus: (1) insulin dependent or Type 1 diabetes (a.k.a., Juvenile Diabetes, Brittle Diabetes, Insulin Dependent Diabetes Mellitus (IDDM)) and (2) non-insulin-dependent or Type II diabetes (a.k.a., NIDDM). Type 1 diabetes develops most often in young people but can appear in adults. Type 2 diabetes develops most often in middle aged and older adults, but can appear in young people. Diabetes is a disease derived from multiple causative factors and characterized by elevated levels of plasma glucose (hyperglycemia) in the fasting state or after administration of glucose during an oral glucose tolerance test.
Type 1 diabetes is an autoimmune disease condition characterized by high blood glucose levels caused by a total lack of insulin, i.e., a complete loss of pancreatic β-cell function and mass. Type 1 diabetes occurs when a person's immune system attacks the insulin producing β-cells in the pancreas and destroys them. It is believed that the Interleukin 12 (IL-12) family of cytokines and downstream activation of Signal Transducers and Activators of Transcription (STAT) family members, e.g., STAT-4, which are believed to be regulators of T cell differentiation involved in immune responses, play a major role in the processes that lead to autoimmune β-cell destruction. The pancreas then produces little or no insulin. The most common Type 1 diabetes symptoms experienced include excessive thirst (polydipsia), frequent urination (polyuria), extreme hunger (polyphagia), extreme fatigue, and weight loss. These symptoms are caused by hyperglycemia and a breakdown of body fats. Persons diagnosed with Type 1 diabetes typically exhibit blood sugar levels over 300 mg and ketones present in their urine. Restoration of β-cell mass and insulin production can fully reverse the diabetic state. Evidence suggests that people with long standing Type 1 diabetes have β-cells that continue to form but are undesirably destroyed by continued autoimmune destruction. Therefore, pharmaceutical compositions and methods for arresting autoimmune β-cell damage would provide an effective way to restore normal β-cell mass levels and reverse or cure Type 1 diabetes.
LADA is a newly recognized subset of Type 1 diabetes and is thought to account for up to 10%-20% of all cases of diabetes. LADA is often present in people initially diagnosed with Type 2 diabetes. Although it has characteristics similar to adult onset type 1 diabetes, the beta-cell destruction is considered to be less aggressive in its progression.
Type 2 diabetes results from a combination of insulin resistance and impaired insulin secretion but ultimately many people with Type 2 diabetes show markedly reduced pancreatic β-cell mass and function which, in turn, causes Type 2 diabetic persons to have a “relative” deficiency of insulin because pancreatic β-cells are producing some insulin, but the insulin is either too little or isn't working properly to adequately allow glucose into cells to produce energy. Recent autopsy studies have shown clear evidence of ongoing β-cell death (apoptosis) in people with Type 2 diabetes. Therefore, therapeutic approaches to arrest β-cell death could provide a significant treatment for reversing or curing Type 2 diabetes.
Uncontrolled Type 2 diabetes leads to excess glucose in the blood, resulting in hyperglycemia, or high blood sugar. A person with Type 2 diabetes experiences fatigue, increased thirst, frequent urination, dry, itchy skin, blurred vision, slow healing cuts or sores, more infections than usual, numbness and tingling in feet. Without treatment, a person with Type 2 diabetes will become dehydrated and develop a dangerously low blood volume. If Type 2 diabetes remains uncontrolled for a long period of time, more serious symptoms may result, including severe hyperglycemia (blood sugar over 600 mg) lethargy, confusion, shock, and ultimately “hyperosmolar hyperglycemic non-ketotic coma.” Persistent or uncontrolled hyperglycemia is associated with increased and premature morbidity and mortality. As such, therapeutic control of glucose homeostasis, lipid metabolism, obesity, and hypertension are critically important in the clinical management and treatment of diabetes mellitus.
The object of diabetes treatments is to prevent the occurrence of the above-mentioned chronic complications, slow disease progression by improving hyperglycemic status, or reversing/curing it. Conventional methods for treating diabetes have included administration of fluids and insulin in the case of Type I diabetes and administration of various hypoglycemic agents in the case of Type II diabetes. Hypoglycemic agents such as insulin preparations, insulin secretagogues, insulin sensitizers and α-glucosidase inhibitors have been widely applied as the method for the clinical treatment. Examples include acarbose (PrecoseJ), glimeprimide (AmarylJ), metformin (Glucophage7), nateglinide (Starlix7), pioglitazone (Actos7), repaglinide (PrandinJ), rosiglitazone (Avandia7), sulfonylureas, Orlistat (Xenical7), exenatide (Byetta), and the like. Many of the known hypoglycemic agents, however, exhibit undesirable side effects and are toxic in certain cases. For example, in the case of the diabetic patients with seriously lowered pancreatic insulin secretion, effectiveness of insulin secretagogues and insulin sensitizers is diminished. Similarly, in the case of the diabetic patients whose insulin resistance is significantly high, effectiveness of insulin preparations and insulin secretagogues is diminished.
In principle, diabetes mellitus could be “cured” by a successful transplant of the tissue containing cells that secrete or produce insulin, i.e., the islets of Langerhans. Transplantation of insulin producing cells (a.k.a., islets) has been tried as a method to reverse or cure Type 1 diabetes, but there are significant risks associated with the surgery and with the toxic immunosuppression type drugs that need to be taken to prevent or mitigate allograft rejection and autoimmune reoccurrence. Immunosuppression drugs act by reducing the activity of a recipient's immune system so that the transplanted insulin producing cells are not rejected. Such immunosuppression, however, entails substantial risks and there are considerable difficulties attendant in minimizing the antigenic differences (matching) between a donor and a recipient that increases the costs and reduces the availability of this mode of therapy. In addition, conventional immunosuppression is generally not successful in enabling islet transplantation. Moreover, there are over 1 million people with Type 1 diabetes in the United States today, but the supply of cadaveric pancreatic tissue for islets is limited. For instance, only 6,000 organs are available per year and 2 or 3 organs are needed to provide enough islets to reverse Type 1 diabetes in one person. Therefore, providing a new source of functioning (insulin producing) β-cells is urgently needed. In addition, if a diabetic patient's own cells (pancreatic or other cell types) could be genetically engineered or induced to grow and differentiate into functioning β-cells, then there would be little or no need to use toxic anti-rejection medications. As previously mentioned, there continues to be the capacity for new β-cell formation in people with Type 1 diabetes. However, continued autoimmunity leads to active destruction of any newly formed or transplanted β-cells. Development of new immunomodulating agents would provide a new way to fully reverse β-cell disfunction in Type 1 diabetes without the need for islet cell transplantation or toxic anti-rejection immunosuppressants. Further, the combination therapy approach provided by a preferred embodiment of present invention would be a major improvement in cellular replacement therapy by reducing the amount of transplanted cells needed to reverse or cure Type 1 diabetes, facilitating the increase viability and growth of insulin producing cells, thereby improving success rates.
Glucagon-Like Peptide (GLP-1) and Gastric Inhibitory Polypeptide (GIP)
Incretins are intestinal hormones released after meal ingestion that stimulate insulin secretion. GLP-1 is a 300-amino-acid (peptide) incretin synthesized in the small and large intestine by the L-type cells of the gastroenteropancreatic endocrine system and is released in response to food ingestion. GLP-1 enhances glucose-stimulated postprandial insulin secretion, stimulates insulin gene expression and proinsulin biosynthesis, inhibits pancreatic glucagons release, gastric emptying, and acid secretion. GIP is another insulin releasing hormone secreted from endocrine cells in the intestinal tract in response to food intake. Together with autonomic nerves, GLP-1 and GIP play a vital supporting role to the pancreatic islets in the control of blood glucose homeostasis and nutrient metabolism.
GLP-1 shows potent insulinotropic action in both diabetic and nondiabetics. GLP-1 causes expansion of beta-cell mass via proliferation of insulin-producing cells. GLP-1 shows an ability to stimulate β-cell neogenesis in streptozotocin (STZ)-treated newborn rats, resulting in persistent improvement of glucose homeostasis to adult age. GLP-1 induces differentiation of islet duodenal homeobox-1-positive pancreatic ductal cells into insulin-secreting cells by enhancing expression of transcription factors PDX-1 and HNF3. GLP-1 has been shown to promote functional maturation of fetal porcine β-cells and islet cell growth in a Type 2 diabetic rat model. Cloning and functional expression of GLP-1 receptors are completed in human islets. GLP-1 receptor signaling directly modifies the susceptibility of β-cells to apoptotic injury that may be the potential mechanism linking to preservation and enhancement of β-cell mass and function. GLP-1 receptor signaling, however, does not seem essential for glucose-stimulated insulin secretion, as shown in GPL-1 receptor knockout mice, which suggests that the functional signaling of GLP-1 in β-cells may be in addition to the one initiated by glucose.
GLP-1 has been studied as a potential drug for the management of diabetes for two reasons: (i) its effect on β-cell growth; and (ii) its insulin-stimulating effect with minimal risk of hypoglycemia and absence of effect on insulin action in non-diabetic humans. In limited clinical trails, GPL-1 is effective in treating Type 2 diabetic patients, showing a significant improvement in postprandial glycemic control and normalization of fasting hyperglycemia due to its ability of insulinotropic activity.
GIP is released from intestinal endocrine K-cells into the bloodstream following ingestion of carbohydrate, protein and particularly fat. GIP's major physiological role is generally believed to be that of an incretin hormone that targets pancreatic islets to enhance insulin secretion and help reduce postprandial hyperglycemia. GIP acts through binding to specific G-protein coupled GIP receptors located on pancreatic beta-cells (Wheeler, M. B. et al., 1995, Endocrinology 136:4629-4639). GIP has been shown to stimulate β-cell proliferation synergistically with glucose in the islet INS-1 cell line, in association with induction of MAPK and PI 3-kinase. Similarly, GIP exerts anti-apoptotic actions in studies using INS-1 β-cells. Like glucagon-like peptide-1 (GLP-1), the ability to stimulate insulin secretion plus other potentially beneficial actions on pancreatic beta-cell growth and differentiation have led to much interest in using GLP-1 or GIP and analogs thereof for the treatment of type 2 diabetes.
Neither, GLP-1 nor GIP, however, appear suitable for therapeutic use in chronic disorders, such as Type 2 diabetes because GLP-1 and GIP are rapidly cleared from blood circulation (half life of about 1.5 min.) by the ubiquitous enzyme dipeptidyl peptidase-IV (DPP-IV). Exogenously administered GLP-1 is also rapidly degraded. This metabolic instability limits the therapeutic potential of native GLP-1 and GIP.
Exendin-4 (Ex-4)
As an analog of GLP-1, Ex-4 was first isolated from the salivary secretions of a South American lizard known as the Gila monster (Heloderma suspectum). Ex-4 consists 39-amino acids with 53% structural homology to mammalian GLP-1. Ex-4 is capable of binding to both human and rat GLP-1 receptors and shows similar pharmacological and biological properties of GLP-1. As a more potent agent than GLP-1, Ex-4 is strongly capable of increasing β-cell mass by enhancing both cell replication and neogenesis, and by inhibiting the apoptosis of β-cells.
In spite of similarities, Ex-4 differs from GLP-1: (i) Ex-4 is resistant to DPP-IV cleavage, resulting in a long-lasting biological function that is potentially suitable for therapeutic use; (ii) Ex-4 has greater insulinotropic efficacy; and (iii) although both GLP-1 and Ex-4 have similar effects to augment insulin-stimulated glucose uptake and metabolism in skeletal muscle, Ex-4 also increases glucose uptake in adipocytes. Ex-4 may also use different signaling pathways, possibly through a receptor other than the GLP-1 receptor. This may render Ex-4 more effective in reducing blood glucose by simultaneously stimulating β-cell insulin secretion and increasing glucose utilization in both skeletal muscle and fat tissue. Ex-4 has also been studied for treatment of Type 2 diabetes, as an additive to existing treatments (such as mefformin and/or sulfonylurea) to control hyperglycemia in Type 2 diabetic patients. An injectable synthetic form of Ex-4 (Byetta® (exenatide) sold by Amylin Pharmaceuticals, Inc.) has been recently approved for use in treating Type 2 diabetes as an adjunctive therapy to improve blood sugar control.
A study recently showed that Ex-4, along with anti-lymphocyte serum (ALS), reversed hyperglycemia in previously overt diabetic NOD (Non-Obese Diabetic) mice. In this study, GLP-1 alone showed no effect to hyperglycemia in NOD mice, indicating that controlling auto-activated lymphocytes by ALS was required to achieve remission of euglycemia. However, ALS is a potent immunosuppressant that causes general dysfunction in all types of lymphocytes. Long-term use of ALS has been known to lead to the risk of tumorigenesis and other severe infectious diseases due to general immune deficiency. Therefore, ALS and other immunosuppressant drugs have not been shown to be clinically useful in treating diabetes.
Accordingly, there remains a need for more effective pharmaceutical compositions and methods that utilize immunomodulating agents alone as monotherapy or in combination with a β-cell growth and/or differentiating factor to restore normal β-cell mass and/or function in subjects suffering from diabetes.